(a) Hypothesis: Upon activation of polycomb target genes, PRC2 switches from binding chromatin to binding nascent RNA.
(b) SUZ12, H3K27me3 and total H3 chromatin occupancy (with IgG control) at Adcy7, Sorcs2 and Actb before and after HRasV12 expression (mean and S.D., n=3 independent ChIPs. Adcy7 SUZ12 P=0.0051, H3K27me3 P=2.1x10-4. Sorcs2 SUZ12 P=0.0041, H3K27me3 P=4.9x10-4, Welch’s one-tailed t-test).
(c) SUZ12 binding to Adcy7, Sorcs2 and Actb nascent pre-mRNA before and after HRasV12 expression, measured by RIP-qPCR with and without UV-crosslinking of cells (mean and S.D., n=3 independent RIPs. Adcy7 SUZ12 +/-Ras P=0.011, Sorcs2 SUZ12 +/-Ras P=0.0019, Welch’s one-tailed t-test).
(d) Change in HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy at Adcy7 and Sorcs2 after dox-mediated induction of HA-dCas9 expression in cells containing sgRNA specific for Adcy7 (mean and S.D., n=3 independent dox inductions. Adcy7 G-tract RNA: SUZ12 P=0.014, H3K27me3 P=0.024, Welch’s one-tailed t-test).
(e) As (d), except in cells containing sgRNA specific for Sorcs2. Sorcs2 G-tract RNA: SUZ12 P=0.017, H3K27me3 P=2.9x10-4, Welch’s one-tailed t-test).